U.S. patent application number 12/830876 was filed with the patent office on 2011-01-13 for microfluidic device adapted for post-centrifugation use with selective sample extraction and methods for its use.
Invention is credited to Gary P. Durack.
Application Number | 20110008818 12/830876 |
Document ID | / |
Family ID | 43427769 |
Filed Date | 2011-01-13 |
United States Patent
Application |
20110008818 |
Kind Code |
A1 |
Durack; Gary P. |
January 13, 2011 |
MICROFLUIDIC DEVICE ADAPTED FOR POST-CENTRIFUGATION USE WITH
SELECTIVE SAMPLE EXTRACTION AND METHODS FOR ITS USE
Abstract
The present disclosure relates to microfluidic devices adapted
for post-centrifugation use with selective sample extraction, and
methods for their use. Certain embodiments make use of a
dye-selective sample extraction. Other embodiments make use of a
geographically-selective sample extraction. Other embodiments are
also disclosed.
Inventors: |
Durack; Gary P.; (Urbana,
IL) |
Correspondence
Address: |
Woodard, Emhardt, Moriarty, McNett & Henry LLP;Sony Corporation
111 Monument Circle, Suite 3700
Indianapolis
IN
46204-5137
US
|
Family ID: |
43427769 |
Appl. No.: |
12/830876 |
Filed: |
July 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61223412 |
Jul 7, 2009 |
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61223413 |
Jul 7, 2009 |
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Current U.S.
Class: |
435/29 ;
435/287.1 |
Current CPC
Class: |
B01L 2300/087 20130101;
B01L 2200/10 20130101; B01L 3/502738 20130101; B01L 3/502753
20130101; G01N 33/491 20130101; B01L 2300/0864 20130101; G01N
2035/00237 20130101; B01L 2400/0409 20130101; B01L 2400/0688
20130101; B01L 3/502761 20130101; B01L 2200/0605 20130101; B01L
2200/0647 20130101; B01L 2200/0652 20130101; G01N 1/4077 20130101;
B01L 2400/0406 20130101; B01L 2300/0816 20130101; B01L 2300/0867
20130101; B01L 2200/027 20130101; G01N 2015/149 20130101 |
Class at
Publication: |
435/29 ;
435/287.1 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12M 1/34 20060101 C12M001/34 |
Claims
1. A method of analyzing a sample fluid in a microfluidic cytometry
system, the method comprising the steps of: a) supplying a sample
to a sample well in a microfluidic device; b) centrifuging the
microfluidic device to create a first sample layer and a second
sample layer within said sample well; c) extracting first fluid
from said first sample layer; and d) analyzing said extracted first
fluid using cytometry while said extracted first fluid is in the
microfluidic device.
2. The method of claim 1, further comprising the steps of: e)
sorting a first portion of said extracted first fluid into a first
well based upon results obtained in step (d); and f) sorting a
second portion of said extracted first fluid into a second well
based upon results obtained in step (d).
3. The method of claim 2, wherein said sorting at steps (e) and (f)
is selected from the group consisting of: greater than a binary
sort, greater than a tertiary sort, greater than a quaternary
sort.
4. The method of claim 2, wherein said sorting at steps (e) and (f)
employs a predetermined number of microfluidic channels, said
predetermined number selected from the group consisting of: at
least five channels, at least ten channels, at least 100 channels,
and at least 256 channels.
5. The method of claim 2, further comprising the steps of: g)
extracting second fluid from said second sample layer; and h)
analyzing said extracted second fluid using cytometry while said
extracted second fluid is in the microfluidic device.
6. The method of claim 5, further comprising the step of: i)
sorting a third portion of said extracted second fluid into a third
well based upon results obtained in step (h).
7. The method of claim 5, wherein: said extracted first fluid is
extracted from said sample well at a first location; and said
extracted second fluid is extracted from said sample well at a
second location, said second location being different than said
first location.
8. The method of claim 1, further comprising the step of: e) adding
a chemical additive to said sample well prior to performing step
(a).
9. The method of claim 1, further comprising the step of: e) adding
Ficoll-Hypaque to said sample well prior to performing step
(a).
10. The method of claim 1, further comprising the step of: e)
adding a dye to said sample well prior to performing step (a).
11. The method of claim 1, wherein step (a) further comprises
supplying a sample of human blood to a sample well on a
microfluidic device
12. A microfluidic device, comprising: a sample well having a
sample input port for receiving a sample into the microfluidic
device, said sample well having a first end and a second end; and a
sample output port in fluid communication with said sample well,
said sample output port located between said first end and said
second end; wherein sample may be withdrawn through said sample
output port to form a withdrawn sample and said withdrawn sample is
not withdrawn at said first end or at said second end.
13. The microfluidic device of claim 12, further comprising: a
sorting well; a waste well; and a channel coupled to said sorting
well and said waste well; a valve disposed in said channel for
directing fluid flowing in said channel into either said sorting
well or said waste well.
14. The microfluidic device of claim 12, further comprising a
chemical additive disposed in the sample well prior to receiving a
sample.
15. The microfluidic device of claim 14, wherein said chemical
additive comprises Ficoll-Hypaque.
16. The microfluidic device of claim 14, wherein said chemical
additive comprises a dye.
17. A microfluidic device, comprising: a sample well having a
sample input port for receiving a sample into the microfluidic
device, said sample well having a first end and a second end; a
first sample output port in fluid communication with said sample
well, said first sample output port located between said first end
and said second end; and a second sample output port in fluid
communication with said sample well, said second sample output port
located between said first sample output port and said second end;
wherein sample may be withdrawn through said first sample output
port to form a first withdrawn sample and said first withdrawn
sample is not withdrawn at said first end or at said second end;
and wherein sample may be withdrawn through said second sample
output port to form a second withdrawn sample and said second
withdrawn sample is not withdrawn at said first end or at said
second end.
18. The microfluidic device of claim 17, further comprising: a
first sorting well; a second sorting well; a waste well; and a
channel coupled said first sorting well, said second sorting well
and said waste well; a valve disposed in said channel for directing
fluid flowing in said channel into either said first sorting well,
said second sorting well or said waste well.
19. The microfluidic device of claim 17, further comprising a
chemical additive disposed in the sample well prior to receiving a
sample.
20. The microfluidic device of claim 19, wherein said chemical
additive comprises Ficoll-Hypaque.
21. The microfluidic device of claim 19, wherein said chemical
additive comprises a dye.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Patent Application No. 61/223,412, which was filed Jul.
7, 2009. The present application also claims the benefit of U.S.
Provisional Application No. 61/223,413, filed Jul. 7, 2009. Both of
these applications are incorporated herein by reference in their
entireties
TECHNICAL FIELD OF THE INVENTION
[0002] The present disclosure relates generally to microfluidic
cytometry systems and, more particularly, to a microfluidic device
adapted for post-centrifugation use with selective sample
extraction and methods for its use.
BACKGROUND OF THE INVENTION
[0003] Flow cytometry-based cell sorting was first introduced to
the research community more than 20 years ago. It is a technology
that has been widely applied in many areas of life science
research, serving as a critical tool for those working in fields
such as genetics, immunology, molecular biology and environmental
science. Unlike bulk cell separation techniques such as
immuno-panning or magnetic column separation, flow cytometry-based
cell sorting instruments measure, classify and then sort individual
cells or particles serially at rates of several thousand cells per
second or higher. This rapid "one-by-one" processing of single
cells has made flow cytometry a unique and valuable tool for
extracting highly pure sub-populations of cells from otherwise
heterogeneous cell suspensions.
[0004] Cells targeted for sorting are usually labeled in some
manner with a fluorescent material. The fluorescent probes bound to
a cell emit fluorescent light as the cell passes through a tightly
focused, high intensity, light beam (typically a laser beam). A
computer records emission intensities for each cell. These data are
then used to classify each cell for specific sorting operations.
Flow cytometry-based cell sorting has been successfully applied to
hundreds of cell types, cell constituents and microorganisms, as
well as many types of inorganic particles of comparable size.
[0005] Flow cytometers are also applied widely for rapidly
analyzing heterogeneous cell suspensions to identify constituent
sub-populations. Examples of the many applications where flow
cytometry cell sorting is finding use include isolation of rare
populations of immune system cells for AIDS research, isolation of
genetically atypical cells for cancer research, isolation of
specific chromosomes for genetic studies, and isolation of various
species of microorganisms for environmental studies. For example,
fluorescently labeled monoclonal antibodies are often used as
"markers" to identify immune cells such as T lymphocytes and B
lymphocytes, clinical laboratories routinely use this technology to
count the number of "CD4 positive" T cells in HIV infected
patients, and they also use this technology to identify cells
associated with a variety of leukemia and lymphoma cancers.
[0006] Recently, two areas of interest are moving cell sorting
towards clinical, patient care applications, rather than strictly
research applications. First is the move away from chemical
pharmaceutical development to the development of
biopharmaceuticals. For example, the majority of novel cancer
therapies are now biologics containing proteins or peptides. These
include a class of antibody-based cancer therapeutics.
Cytometry-based cell sorters can play a vital role in the
identification, development, purification and, ultimately,
production of these products.
[0007] There is also a move toward the use of cell replacement
therapy for patient care. Much of the current interest in stem
cells revolves around a new area of medicine often referred to as
regenerative therapy or regenerative medicine. These therapies may
often require that large numbers of relatively rare cells be
isolated from sample patient tissue. For example, adult stem cells
may be isolated from bone marrow or adipose tissue and ultimately
used as part of a re-infusion back into the patient from whom they
were removed. Cytometry lends itself very well to such
therapies.
[0008] There are two basic types of cell sorters in wide use today.
They are the "droplet cell sorter" and the "fluid switching cell
sorter." The droplet cell sorter utilizes micro-droplets as
containers to transport selected cells to a collection vessel. The
micro-droplets are formed by coupling ultrasonic energy to a
jetting stream. Droplets containing cells selected for sorting are
then electrostatically steered to the desired location. This is a
very efficient process, allowing as many as 90,000 cells per second
to be sorted from a single stream, limited primarily by the
frequency of droplet generation and the time required for
illumination.
[0009] A detailed description of a prior art flow cytometry system
is given in United States Published Patent Application No. US
2005/0112541 A1 to Durack et al.
[0010] Droplet cell sorters, however, are not particularly biosafe.
Aerosols generated as part of the droplet formation process can
carry biohazardous materials. Because of this, biosafe droplet cell
sorters have been developed that are contained within a biosafety
cabinet so that they may operate within an essentially closed
environment. Unfortunately, this type of system does not lend
itself to the sterility and operator protection required for
routine sorting of patient samples in a clinical environment.
[0011] The second type of flow cytometry-based cell sorter is the
fluid switching cell sorter. Most fluid switching cell sorters
utilize a piezoelectric device to drive a mechanical system which
diverts a segment of the flowing sample stream into a collection
vessel. Compared to droplet cell sorters, fluid switching cell
sorters have a lower maximum cell sorting rate due to the cycle
time of the mechanical system used to divert the sample stream.
This cycle time, the time between initial sample diversion and when
stable non-sorted flow is restored, is typically significantly
greater than the period of a droplet generator on a droplet cell
sorter. This longer cycle time limits fluid switching cell sorters
to processing rates of several hundred cells per second. For the
same reason, the stream segment switched by a fluid cell sorter is
usually at least ten times the volume of a single micro-drop from a
droplet generator. This results in a correspondingly lower
concentration of cells in the fluid switching sorter's collection
vessel as compared to a droplet sorter's collection vessel.
[0012] Newer generation microfluidics technologies offer great
promise for improving the efficiency of fluid switching devices and
providing cell sorting capability on a chip similar in concept to
an electronic integrated circuit. Many microfluidic systems have
been demonstrated that can successfully sort cells from
heterogeneous cell populations. They have the advantages of being
completely self-contained, easy to sterilize, and can be
manufactured on sufficient scales (with the resulting manufacturing
efficiencies) to be considered a disposable part.
[0013] A generic microfluidic device is illustrated in FIG. 1 and
indicated generally at 10. The microfluidic device 10 comprises a
substrate 12 having a fluid flow channel 14 formed therein by any
convenient process as is known in the art. The substrate 12 may be
formed from glass, plastic or any other convenient material, and
may be substantially transparent or substantially transparent in a
portion thereof. In certain embodiments, the substrate 12 is
injection molded. In certain embodiments, the substrate 12
comprises industrial plastic such as a Cyclo Olefin Polymer (COP)
material, or other plastic. As a result, the substrate 12 is
transparent such that a cytometry optics module can analyze the
sample fluid stream as described further below. In one embodiment,
the microfluidic device 10 is disposable.
[0014] The substrate 12 further has three ports 16, 18 and 20
coupled thereto. Port 16 is an inlet port for a sheath fluid. Port
16 has a central axial passage that is in fluid communication with
a fluid flow channel 22 that joins fluid flow channel 14 such that
sheath fluid entering port 16 from an external supply (not shown)
will enter fluid flow channel 22 and then flow into fluid flow
channel 14. The sheath fluid supply may be attached to the port 16
by any convenient coupling mechanism as is known to those skilled
in the art. In one embodiment, the sheath fluid comprises a buffer
or buffered solution. For example, the sheath fluid comprises 0.96%
Dulbecco's phosphate buffered saline (w/v), 0.1% BSA (w/v), in
water at a pH of about 7.0.
[0015] Port 18 also has a central axial passage that is in fluid
communication with a fluid flow channel 14 through a sample
injection tube 24. Sample injection tube 24 is positioned to be
coaxial with the longitudinal axis of the fluid flow channel 14.
Injection of a liquid sample of cells into port 18 while sheath
fluid is being injected into port 16 will therefore result in the
cells flowing through fluid flow channel 14 surrounded by the
sheath fluid. The dimensions and configuration of the fluid flow
channels 14 and 22, as well as the sample injection tube 24 are
chosen so that the sheath/sample fluid will exhibit laminar flow as
it travels through the device 10, as is known in the art. Port 20
is coupled to the terminal end of the fluid flow channel 14 so that
the sheath/sample fluid may be removed from the microfluidic device
10.
[0016] While the sheath/sample fluid is flowing through the fluid
flow channel 14, it may be analyzed using cytometry techniques by
shining an illumination source through the substrate 12 and into
the fluid flow channel 14 at some point between the sample
injection tube 24 and the outlet port 20. Additionally, the
microfluidic device 10 could be modified to provide for a cell
sorting operation, as is known in the art.
[0017] Although basic microfluidic devices similar to that
described hereinabove have been demonstrated to work well, there is
a need in the prior art for improvements to cytometry systems
employing microfluidic devices. The present invention is directed
to meeting this need.
SUMMARY OF THE DISCLOSURE
[0018] The present disclosure relates to microfluidic devices
adapted for post-centrifugation use with selective sample
extraction, and methods for their use. Certain embodiments make use
of a dye-selective sample extraction. Other embodiments make use of
a geographically-selective sample extraction.
[0019] In certain embodiments, a method of analyzing a sample fluid
in a microfluidic cytometry system is disclosed, the method
comprising the steps of a) supplying a sample to a sample well in a
microfluidic device; b) centrifuging the microfluidic device to
create a first sample layer and a second sample layer within said
sample well; c) extracting first fluid from said first sample
layer; and d) analyzing said extracted first fluid using cytometry
while said extracted first fluid is in the microfluidic device.
[0020] In other embodiments, a microfluidic device is disclosed,
comprising a sample well having a sample input port for receiving a
sample into the microfluidic device, said sample well having a
first end and a second end, and a sample output port in fluid
communication with said sample well, said sample output port
located between said first end and said second end, wherein sample
may be withdrawn through said sample output port to form a
withdrawn sample and said withdrawn sample is not withdrawn at said
first end or at said second end.
[0021] In further embodiments, a microfluidic device is disclosed,
comprising a sample well having a sample input port for receiving a
sample into the microfluidic device, said sample well having a
first end and a second end, a first sample output port in fluid
communication with said sample well, said first sample output port
located between said first end and said second end, and a second
sample output port in fluid communication with said sample well,
said second sample output port located between said first sample
output port and said second end, wherein sample may be withdrawn
through said first sample output port to form a first withdrawn
sample and said first withdrawn sample is not withdrawn at said
first end or at said second end, and wherein sample may be
withdrawn through said second sample output port to form a second
withdrawn sample and said second withdrawn sample is not withdrawn
at said first end or at said second end.
[0022] Other embodiments are also disclosed.
DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a perspective view of a prior art microfluidic
device.
[0024] FIG. 2 is a schematic front view of a microfluidic device
according to a first embodiment of the present disclosure.
[0025] FIG. 3 is a schematic front view of a microfluidic device
according to a second embodiment of the present disclosure.
[0026] FIG. 4 is a schematic front view of a microfluidic device
according to a third embodiment of the present disclosure.
[0027] FIG. 5 is a schematic front view of a microfluidic device
according to a fourth embodiment of the present disclosure.
DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS
[0028] For the purposes of promoting an understanding of the
principles of the disclosure, reference will now be made to the
embodiments illustrated in the drawings and specific language will
be used to describe the same. It will nevertheless be understood
that no limitation of the scope of the disclosure is thereby
intended, such alterations and further modifications in the
illustrated device, and such further applications of the principles
of the disclosure as illustrated therein are contemplated as would
normally occur to one skilled in the art to which the disclosure
relates.
[0029] The present disclosure is generally directed to systems for
the separation and/or analysis of a biological sample on a
microfluidic device using cytometry (such as flow cytometry or
image cytometry). To increase the efficiency of cytometry
operations, it is desirable to start with a sample containing
mostly those types of cells that are desired to be individually
studied or isolated. One method known in the art is to subject the
sample to centrifugation prior to cytometry analysis. After
centrifugation, the sample components will be separated into
layers. Due to factors such as mass, density and specific gravity,
the desired component, or cell population, can then be more easily
extracted. This process is common laboratory practice and many
common sample containers and centrifuge devices are commercially
available for this purpose. However, this procedure introduces the
possibility that the sample layers will become remixed during the
transfer from the initial collection vessel to the microfluidic
device. By adapting the microfluidic device itself to be suitable
for containing the sample during the centrifugation process, the
possibility of layer mixing after centrifugation and prior to
analysis by the cytometer is reduced. This also eliminates the need
for multiple containers and reduces the possibility of outside
contaminates being introduced into the sample (or potentially
harmful sample components being released into the outside
environment). Since the microfluidic device can be manufactured to
be sterile (e.g. exposed to gamma irradiation), the entire process
of centrifugation, cytometry measurement, and sorted fraction
collection can be conducted in a closed, sterile environment. Most
importantly, the centrifugation process and transfer of material
among containers can introduce loss of 10% or more of the cells.
When low cell numbers are involved this loss is intolerable, in
fact it is impossible to apply even microcentrifugation in many
cases where sample volume is low.
Dye-Selective Sample Extraction
[0030] In one embodiment, FIG. 2 illustrates a device 200 which is
configured to achieve the separation of biological components
contained within a sample. The device 200 includes a sample well
202 for receiving a sample 204 from an external source (not shown)
via port 206. In certain embodiments, the sample well 202 is
elongated to facilitate gradient separation of the sample
components when the device 200 is placed in a centrifuge. Various
chemical additives known in the art and commonly used to facilitate
the process of separation based on the density of sample
constituents, such as Ficoll-Hypaque to name just one non-limiting
example, may be added to the sample 204 to increase the separation
effect. In certain embodiments, such chemical additives may be
added to the sample well 202 during manufacturing, eliminating the
need for the user to add them during the sampling process. In
addition, in certain embodiments a dye may be added to the
Ficoll-Hypaque to provide a color distinction between the layers as
described hereinbelow.
[0031] As will be appreciated by those skilled in the art, the
microfluidic device may be subjected to the forces of
centrifugation in either direction along the longitudinal axis of
the sample well 202. With the Ficoll-Hypaque method, the cells are
layered on to the Ficoll-Hypaque solution, so that the cells are
"on top" relative to the force of centrifugation. In other
embodiments and other additives, the entire situation might be
inverted. By using the microfluidic device as the container for the
sample before analysis, during centrifugation, during analysis
and/or after sorting, the user may move the small sample from a
centrifuge in one part of the laboratory to a cytometer somewhere
else, and then after analysis the sample may be transported to a
third location, all in a sterile environment.
[0032] Channel 208 is connected via port 210 at one end of sample
well 202 to retrieve the desired sample component after
centrifugation. As one non-limiting example, human blood may be
injected into the sample well 202 after which the device 200 is
subjected to centrifugation to separate the mononuclear cells 212,
i.e. lymphocytes and monocytes, from the larger granulocytic cells
214 and red blood cells 216. After centrifugation, the port 210 may
be opened to allow the sample 204 to flow through channel 208 to a
cytometry analysis section 218 within device 200 for further
cytometric analysis and/or separation. For example, the cytometry
analysis section 218 may image the cells in the flow channel prior
to switching element 223 and then divert a subset of the
mononuclear cells previously isolated, by identifying them using
image or common fluorescence-based methods, and then exercising
appropriate control of a switching element 223, into an extraction
well 222 and undesirable cells into a waste well 224. In certain
embodiments, the wells 222 and 224 communicate with ports (not
shown) to allow extraction of their contents from the microfluidic
device 200. The specific analysis and/or sorting performed in
analysis section 218 is not critical to the present disclosure and
may be performed in a variety of different manners in different
embodiments.
[0033] In certain embodiments, the sorting performed by the
cytometry analysis section 218 is greater than a binary sort. In
other embodiments, the sorting performed by the cytometry analysis
section 218 is greater than a tertiary sort. In other embodiments,
the sorting performed by the cytometry analysis section 218 is
greater than a quartnery sort. In executing the sort, certain
embodiments comprise at least five channels. Other embodiments
comprise at least ten channels. Other embodiments comprise at least
100 channels. Other embodiments comprise at least 256 channels.
[0034] The flow through channel 208 may be initiated by capillary
action or other microfluidic flow means known in the art. Such
methods may require that additional channels or features (not
shown) be included in the microfluidic device. In one embodiment,
sample cells are added to the sample well 202 first and then the
Ficoll-Hypaque solution is added. The microfluidic device is then
subjected to centrifugation with the applied force being toward the
port 206. As the sample flows through the channel 208, the lighter
mononuclear cells 212 will be drawn off first. The cytometry
analysis and sorting section 218 is programmed to close the port
210 or, in alternative embodiments, a valve 220, upon the detection
by analysis section 218 of the particular dye contained within the
Ficoll-Hypaque solution. In this case the dye will be in the same
layer as the plasma-Ficoll interface which is just below the layer
of lymphocytes. The granulocytic cells 214 and red blood cells 216
would be held below the plasma-Ficall interface layer and,
therefore, the dye. This allows the full amount of mononuclear
cells 212 contained in the sample 204 to be extracted while still
rejecting the undesired components within the gradient separation.
In a second example, necrotic cells can easily be separated without
any chemical additives due to their higher density. In addition to
blood, the present disclosure contemplates that other types of
biological or chemical samples may be analyzed in this manner, it
being understood that the present disclosure is useful with any
sample that can be separated by centrifugation.
[0035] When dye is added to the sample solution, it must be done in
such a way that the dye ends up in the correct layer after
centrifugation. In certain embodiments, the dye may be designed to
have the correct density to achieve this. In other embodiments, the
dye can be bonded at the molecular level with the Ficall-Hypaque
(or other chemical additive) in a manner that does not negatively
impact the specific gravity of the Ficoll-Hypaque.
[0036] In other embodiments, various density gradient chemicals,
each with their own specific gravity and/or dye color, can be used
to detect and extract individual components from a sample. For
example, as shown in FIG. 3, the cytometry analysis section 318 may
be configured to divert sorted cells or particles (such as viral
particles) from a first sample component into a first extraction
well 322 until the detection of a first colored dye corresponding
to the density gradient chemical layer (which holds a second sample
component 304). The device 300 is similar to the device 200, and
like reference designators are used to designate like components.
After centrifugation, the cytometry analysis section 318 will
adjust the analysis parameters to account for the new type of cells
being analyzed and divert sorted cells into a second extraction
well 326. Upon the detection of a second colored dye solution
(which holds a third sample component 306), the cytometry analysis
section 318 will then adjust the analysis parameters to account for
the new type of cells being analyzed and divert all remaining cells
into the waste well 324 and/or close valve 220 to cease sample
flow. In certain embodiments, the wells 322, 324 and 326
communicate with ports (not shown) to allow extraction of their
contents from the microfluidic device 300. The specific analysis
and/or sorting performed in analysis section 318 is not critical to
the present disclosure and may be performed in a variety of
different manners in different embodiments.
[0037] In certain embodiments, the sorting performed by the
cytometry analysis section 318 is greater than a binary sort. In
other embodiments, the sorting performed by the cytometry analysis
section 318 is greater than a tertiary sort. In other embodiments,
the sorting performed by the cytometry analysis section 318 is
greater than a quartnery sort. In executing the sort, certain
embodiments comprise at least five channels. Other embodiments
comprise at least ten channels. Other embodiments comprise at least
100 channels. Other embodiments comprise at least 256 channels.
[0038] In certain embodiments, the microfluidic devices comprise
two or more pieces. The two pieces are coupled together using any
desirable means such as, by way of non-limiting example, a thermal
bonding process, an ultrasonic welding process or an adhesive
process. In one embodiment the two pieces are halves. In another
embodiment, the two pieces are divided asymmetrically in the plane,
e.g. a planar cover and a piece containing channels. In still other
embodiments, multiple pieces are assembled. Other ways of coupling
the two pieces will be readily apparent to one skilled in the art
depending upon the material used to fabricate the microfluidic
device.
Geographically-Selective Sample Extraction
[0039] FIG. 4 illustrates another embodiment, in which a device 400
which is configured to achieve the separation of biological
components contained within a sample. The device 400 is similar to
the device 200, and like reference designators are used to
designate like components. The device 400 includes a sample well
202 for receiving a sample 204 from an external source (not shown)
via port 206. The sample well 202 is elongated to facilitate
gradient separation of the sample components when the device 400 is
placed in a centrifuge. Various chemical additives known in the
art, such as Ficoll-Hypaque, may be added to the sample 204 to
increase the separation effect as described hereinabove. In certain
embodiments, such chemical additives may be added to the sample
well 202 during manufacturing, eliminating the need for the user to
add them during the sampling process.
[0040] Channel 208 is connected via port 210 at a specific location
along the length of sample well 202 to retrieve the desired sample
component after centrifugation. The exact location of the port 210
is determined by the nature of the sample being processed and the
component desired to be extracted, such that the placement of the
port 210 is designed to be in close physical proximity to the
position of the component desired to be extracted after
centrifugation. This ensures that a maximum volume of the desired
sample component may be extracted from the sample well 202. It will
be appreciated, therefore, that the placement of the port 210 in
the device 400 is made strategically to more precisely align with
the location of the desired sample component than the device 200
illustrated in FIG. 2.
[0041] As one non-limiting example, human blood may be injected
into the sample well 202 after which the device 400 is subjected to
centrifugation to separate the mononuclear cells 212, i.e.
lymphocytes and monocytes, from the larger granulocytic cells 214
and red blood cells 216. After centrifugation, the port 210 may be
opened to allow the sample 204 to flow through channel 208 to a
cytometry analysis section 218 within device 400 for further
cytometric analysis and/or separation. For example, the cytometry
analysis section 218 may divert sorted desirable cells, by
appropriate control of the switching device 223, into an extraction
well 222 and undesirable cells into a waste well 224. In certain
embodiments, undesirable cells may also be expelled from the device
400 through a waste port (not shown). The specific analysis and/or
sorting performed in analysis section 218 is not critical to the
present disclosure.
[0042] The flow through channel 208 may be initiated by capillary
action or other microfluidic pumping means known in the art. As the
sample flows through the channel 208, the lighter mononuclear cells
212 will be drawn off first due to their proximity to the port 210.
In certain embodiments, multiple channels 208 and multiple ports
210 may be connected along the sample well 202 within the region of
the desired sample component (such as mononuclear cells 212) to
facilitate more complete extraction of the desired sample
component. The cytometry analysis section 218 is programmed to
close the port 210 or, in alternative embodiments, a valve 220,
after a certain amount of sample fluid or cells has been extracted.
This amount depends on the volume of the sample 204, the nature of
the sample being processed, and the expected volume of the desired
component layer.
[0043] For simplicity and ease of illustration, the presently
illustrated embodiments show single channels extending between the
components, areas or sections of the illustrated devices. However,
it should be appreciated that the single channels may be
representative of multiple cytometry channels and a variety of
possible configurations of channels as would occur to one skilled
in the art.
[0044] In other embodiments, multiple channels can be used to
extract multiple components from a sample. The addition of two or
more types of Ficoll-Hypaque (or other appropriate additive) can
also be used to create additional layers in the centrifuged sample.
For example, as shown in FIG. 5, a device 500 is provided into
which an analysis sample 202 may be loaded through port 206. By the
use of appropriate additives as discussed herein, the analysis
sample may, for example, be divided into three layers 302, 304 and
306 post-centrifugation. The device 500 is adapted to selectively
extract sample from the layers 302 and 304 by positioning ports
310a and 310b, respectively, in alignment with the layers 302 and
304. Port 310a and or valve 320a may be opened to allow sample 302
to flow through the channel 308a. A cytometry analysis section 318
may be configured to divert sorted cells from the first sample
component 302 into a first extraction well 322 by control of valve
323. Similarly, undesired cells from the first sample component 302
may be diverted to waste well 324 by appropriate control of the
valve 323. Once the cytometry section 318 determines that analysis
of the first sample component 302 is complete, the cytometry
analysis section 318 is programmed to close the port 310a or, in
alternative embodiments, a valve 320a.
[0045] The cytometry analysis section 318 will then adjust the
analysis parameters to account for the new type of cells being
analyzed, open port 310b, allowing fluid from the second sample
component 304 to flow to the cytometry analysis section 318, and
divert sorted cells into a second extraction well 326 by
appropriate control of valve 323. Once the cytometry section 318
determines that analysis of the second sample component 304 is
complete, the cytometry analysis section 318 is programmed to close
the port 310b or, in alternative embodiments, a valve 320b.
[0046] It will be appreciated that by proper location of the ports
310a and 310b, the device 500 may take advantage of the multiple
layers of sample post-centrifugation. Analysis and sorting of the
sample components is made easier by separating the sample
components into layers using centrifugation, and then extracting
sample components in a purer form by means of strategic location of
the extraction ports 310. It will be appreciated by those skilled
in the art that any number of extraction ports may be utilized to
more precisely extract post-centrifugation samples from the
analysis sample.
[0047] While the disclosure has been illustrated and described in
detail in the drawings and foregoing description, the same is to be
considered as illustrative and not restrictive in character, it
being understood that only the preferred embodiments have been
shown and described and that all changes and modifications that
come within the spirit of the disclosure are desired to be
protected.
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